Design, Synthesis, and Structural Characterization of Novel Diazaphenothiazines with 1,2,3-Triazole Substituents as Promising Antiproliferative Agents

A series of novel 1,2,3-triazole-diazphenothiazine hybrids was designed, synthesized, and evaluated for anticancer activity against four selected human tumor cell lines (SNB-19, Caco-2, A549, and MDA-MB231). The majority of the synthesized compounds exhibited significant potent activity against the investigated cell lines. Among them, compounds 1d and 4c showed excellent broad spectrum anticancer activity, with IC50 values ranging from 0.25 to 4.66 μM and 0.25 to 6.25 μM, respectively. The most promising compound 1d, possessing low cytotoxicity against normal human fibroblasts NHFF, was used for gene expression analysis using reverse transcription–quantitative real-time PCR (RT–qPCR). The expression of H3, TP53, CDKN1A, BCL-2, and BAX genes revealed that these compounds inhibited the proliferation in all cells (H3) and activated mitochondrial events of apoptosis (BAX/BCL-2).


Introduction
The phenothiazines were the first synthetic agents to be used successfully in psychiatry as neuroleptics. The properties of this group of heterocyclic compounds was discovered in the 1950s and provoked revolutions in contemporary medicine [1]. Currently, they are also well-known clinically as substances with antibacterial, antiviral, anti-inflammatory, and anti-tuberculosis properties. There have also been numerous reports on the antitumor activity of these drugs [2][3][4][5][6]. Neuroleptic phenothiazines became the foundation for numerous studies on modified phenothiazine systems that show a wide spectrum of biological activities, including: anticancer, antibacterial, antifungal, antiviral, and antiparasitic. This rich scientific material is the subject of numerous publications that can be found in the world literature every year [7][8][9][10][11][12][13][14][15][16].
The identification of the product structure was based on 1 H, 13 C NMR spectra, 2D NMR experiments: COSY and ROESY, and mass spectrometry HRMS.
The crude products of the reactions were separated by column chromatography to obtain pure final derivatives (1-4)b-f with good yields.
On the basis of previous studies, the reaction of 2-propynyl derivatives with organic azides can led to 1,4-and/or 1,5-regioisomers [26,28]. Bearing in mind the literature, using a CuI catalyst, a 1,4 regioisomer is obtained selectively [28,34]. Nevertheless, the structure of the products received had to be unambiguously confirmed. For this reason, the 2D NMR ROESY experiment of compound 1b was carried out ( Figure 1, Table 1, Supplementary Material). For triazole hybrid 1b, protons of the benzylic group (CH 2 N Tr where Tr-triazole ring) intercorrelated with aromatic proton of the 1,2,3-triazole ring and orto-protons of the benzene ring. However, if a substitution product of 1,5-regioisomer 1B was formed in the reaction, intercorrelation between the CH 2 group at the thiazine nitrogen atom (CH 2 N Th where Th-thiazine ring) with the CH 2 group at the triazole ring (CH 2 N Tr ) would be expected but was not observed. On the basis of previous studies, the reaction of 2-propynyl derivatives with organic azides can led to 1,4-and/or 1,5-regioisomers [26,28]. Bearing in mind the literature, using a CuI catalyst, a 1,4 regioisomer is obtained selectively [28,34]. Nevertheless, the structure of the products received had to be unambiguously confirmed. For this reason, the 2D NMR ROESY experiment of compound 1b was carried out ( Figure 1, Table 1, Supplementary Material). For triazole hybrid 1b, protons of the benzylic group (CH2NTr where Tr-triazole ring) intercorrelated with aromatic proton of the 1,2,3triazole ring and orto-protons of the benzene ring. However, if a substitution product of 1,5regioisomer 1B was formed in the reaction, intercorrelation between the CH2 group at the thiazine nitrogen atom (CH2NTh where Th-thiazine ring) with the CH2 group at the triazole ring (CH2NTr) would be expected but was not observed.   This we consider to be proof of the presence of 1,4-disubstituted 1,2,3-triazole ring. The full correlation between the neighbouring protones showed COSY spectra (Table 1, Supplementary Material).

Anticancer Activity
Encouraged by our previous promising results in the field of antiproliferative activity of dipyridothiazines [17][18][19][20][21][22][23][24][25] as well as by the good anticancer activity of the 1,2,3-triazoles [26][27][28][29][30][31][32][33], we decided to combine the biological potentials of dipyridothiazines and triazole moieties, hoping to obtain compounds with better activity. The antiproliferative activity of all 20 new 10-substituted 1,2,3-triazole derivatives of dipyridothiazines was tested in vitro against cancer cell lines: glioblastoma SNB-19, colorectal carcinoma Caco-2, lung cancer A549, and breast cancer MDA-MB231 using the WST-1 assay. Normal human fibroblasts NHDF were used as a control. Cisplatin was used as a positive control to induce cell death. The results of the cytotoxicity studies were summarized in Table 2. The most active derivative in relation to all tumor groups was compound 1d, which structure contained the p-chlorobenzyl substituent (IC 50 = 0.25-4.66 µM). This compound also showed relatively low cytotoxicity in relation to normal fibroblasts NHDF due to the vacancy of the examined controls. This compound showed higher activity compared to cisplatin (IC 50 = 0.60-10.53 µM). In the 1,6-diazaphenothiazine group, derivative 1c with the p-fluorobenzyl substituent was also characterized by high anticancer activity in relation to SNB-19 and MDA-MB231 cell lines (IC 50 = 5.72-7.82 µM). On the other hand derivative 3f (with the phenyltiomethyl substituent in 1,2,3-triazole system) acted selectively towards A549 and MDA-MB231 cell lines. In the 3,6-diazaphenothiazine group, the highest anticancer activity was characterized by 4c derivative with the p-fluorobenzyl substituent (IC 50 = 0.25-6.25 µM). This compound showed high cytotoxicity in relation to all cancer cells lines, however, it also showed high cytotoxicity in relation to normal human fibroblasts (IC 50 = 2.85 µM).
Most of the tested compounds (1-4)b-f were characterized by significant antiproliferative activity in relation to the examined cancer cell lines and relatively weak cytotoxicity with respect to normal human fibroblasts. This activity was largely dependent on the location of nitrogen atoms in isomeric dipyridothiazines as well as on the substituent in the benzene ring of the 1,2,3-tiazole system.

Apoptosis Assay
10-((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-1,6-diazaphenothiazine (1d)-the most active compound-was selected to study the mechanism of anticancer activity using RT-qPCR. In this method the gene transcriptional activities of proliferation marker (H3), cell cycle regulator (TP53 and CDKNIA), and intracellular apoptosis pathway (BCL-2 and BAX) were analyzed. The results obtained on four cancer cell lines are collected in Table 3. It has been known that the growth, metabolism, and eventual death of the cells in the body are controlled by hundreds of genes working together [35][36][37]. The gene encoding histone H3 plays a crucial role in regulation of the expression of the genetic information included in the DNA. Compound 1d strongly changed the mRNA copy number of the histone H3 gene in all cancer lines which has an influence on the modification of the chromatin structure in the cells. It is well known, that cellular stresses such as the DNA damage and oncogene activation can change the expression of the TP53 gene encoding the p53 protein which is called the guardian of the genome of the cell. The p53 protein influences cell cycle arrest by changing the expression of CDKN1A gene encoding the p21 protein. The essential function of the p21 protein is to arrest cell cycle progression by inhibiting the activity of cyclin-dependent kinases [37][38][39].
Compound 1d significantly interfered with the amount of mRNA copies of TP53 in all investigated cancer lines. There was also a strong decline of CDKN1A copies in all cancer cells what suggests the possibility of participation in cell cycle arrest.
In biochemical processes of cells, the p53 protein determines the standard balance between the expression of the proapoptotic BAX gene and the antiapoptotic BCL-2 gene. The balance between these proteins determines whether the cell is undergoing apoptosis or interrupting the process. It is believed that the main mechanism of action of the BCL-2 family of proteins regulates the release of cytochrome c from mitochondria by changing the mitochondrial membrane permeability [40,41].
Compound 1d remarkably reduced the expression of BCL-2 in SNB-19, A549 and MDA-MB231 cancer lines, but there was a slight increase in Caco-2 cell line. On the other hand, a decrease of the expression of BAX was observed in all investigated cancer cell lines.
Analysis of the gene expression ratio BCL-2/BAX in MDA-MB231 cell showed activation of the mitochondrial apoptosis as the internal pathway of cell death. Transcriptional activity of these genes in the SNB-19, Caco-2, and A549 cells suggests a different way of cell death, possibly associated with the external pathway of apoptosis. The presented results indicate the possibility of a two-way mechanism of apoptosis caused by the hybrid of diazaphenothiazine with the 1,2,3-triazole system. However, further research is required to fully confirm this hypothesis.

Chemistry
The standard NMR spectra were recorded on Bruker Avance spectrometers ( 1 H at 600 MHz, 13 C at 150 MHz) in CDCl 3 . Two-dimensional COSY and ROESY, spectra of selected compounds were recorded on a Bruker Avance spectrometer at 600 MHz. The HRMS spectra (EI-electro impact ionisation) were run on a Brucker Impact II. The thin layer chromatography was performed on aluminium oxide 60 F 254 neutral (type E) (Merck 1.05581) with CHCl 3 -EtOH (10:1 v/v) as eluent.

Cell Proliferation and Viability
Antiproliferative effect of the obtained compounds on the cancer cells was determined using the cell proliferation reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). This assay is based on the viable cell's ability to cleave the bright red-colored tetrazolium salt (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) to dark red soluble formazan by cellular enzymes. An increase in the amount of formazan dye formed correlates to the number of metabolically active cells in the culture. The formazan dye produced by metabolically active cells is quantified by a scanning ELISA reader by measuring the absorbance of the dye solution at appropriate wavelengths. The examined cells were exposed to the tested compounds for 72 h at various concentrations between 0.1 µg/mL and 100 µg/mL (prepared initially at concentration of 1 mg/mL in DMSO). The cells were incubated with WST-1 (10 µL) for 1 h and the absorbance of the samples against a background control was measured at 450 nm using a microplate reader with a reference wavelength at 600 nm. The results are expressed as means of at least two independent experiments performed in triplicate. The antiproliferative activity of the tested compound was compared to cisplatin. The IC 50 values (a concentration of a compound that is required for 50% inhibition) were calculated from the dose-response relationship with respect to control.

The RT-qPCR Method
Genes trancriptional activity (H3, TP53, CDKN1A, BCL-2, and BAX) was evaluated by real time RT-qPCR with OPTICON TM DNA Engine (MJ Research, Watertown, MA, USA) and QuantTect ® SYBR ® Green RT-PCR Kit (Quiagen, Valencia, CA). Cells were exposed to compound 4 at a 0.5 µg/mL concentration for 24 h. The RNA extraction was made by using Quick-RNA™ Kit MiniPrep (ZYMO RESEARCH). Total RNA integrity was analysed in 1.2% agarose electrophoresis with added ethidium bromide. The quantity and purity of extracted total RNA were determined by using spectrophotometric analysis with HP845 (Hewlett Packard, Waldbronn, Germany) spectrophotometer. The statistical analysis was performed using the Statistica 8.0 software. All values were expressed as means ± SE.

Conclusions
We report here efficient synthesis of a new isomeric series of novel 1,2,3-triazole-dipyridothiazine hybrids in the 1,3-dipolar cycloadditon reactions. The structure of these new compounds was identified using advanced two-dimensional 1 H and 13 C NMR (COSY, ROESY) spectra. Most of the obtained derivatives of diazaphenothiazines exhibited significant anti-proliferative activities against the human glioblastoma SNB-19, colorectal carcinoma Caco-2, breast cancer MDA-MB-231 and lung cancer A549 cell lines with the IC 50 values < 1 µM and were more potent than cisplatin. The most promising compound 1d was used for gene expression analysis by the RT-qPCR method. The expression of H3, TP53, CDKN1A, BCL-2, and BAX genes revealed that this compound inhibited the proliferation in all cells (H3) and activated mitochondrial events of apoptosis (BCL-2/BAX) in MDA-MB231 cancer cell line. Further in vitro and in vivo studies are required to determine the potential pharmacological use of these dipyrido-1,2,3-triazole hybrids in anticancer therapy.